Evaluation of voriconazole anti-Acanthamoeba polyphaga in vitro activity, rat cornea penetration and efficacy against experimental rat Acanthamoeba keratitis.

Background: Acanthamoeba keratitis (AK) is a sight-threatening infectious disease. Its effective and safe medical therapy remains highly debated. Recently, voriconazole, a monotriazole with noted in vitro activity against a large variety of fungi, has been successfully used both topically and systemically to treat human AK cases. Objectives: To measure anti-Acanthamoeba polyphaga in vitro activity, anti-rat AK efficiency and rat cornea penetration of eye-drop and oral voriconazole. Methods: A. polyphaga was maintained in axenic cultures. In vitro, amoebicidal and cysticidal activities of voriconazole were measured using an XTT assay. AK lesions of Sprague Dawley rats were scored from grade 0 to grade 3. For 21 days, from day 7 post-infection, voriconazole (1% solution) eye drops were instilled or voriconazole was administered by gavage (60 mg/kg/day). After killing, superficial corneal epithelium scrapings were cultured and analysed by PCR, and eye-globe histology was performed. Cornea and plasma concentrations were determined using 2D HPLC separation and tandem MS. Results: In vitro, voriconazole inhibited trophozoite proliferation with an IC50 value of 0.02 mg/L and an IC90 value of 2.86 mg/L; no cysticidal effect was found. In AK rats, eye drops reduced clinical worsening from day 7 to day 14 post-infection and oral voriconazole was not effective. Voriconazole cornea concentrations were directly dependent on the frequency of eye-drop instillations, which resulted in lower plasma concentrations, whilst oral voriconazole resulted in lower cornea concentrations. Conclusions: Present data underline the need for high-frequency eye-drop instillation regimens for efficient AK therapy.

A sensitive LC-MS/MS method for the quantification of regioisomers of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids in human plasma during endothelial stimulation.

Epoxyeicosatrienoic acids (EETs) are vasodilating lipid mediators metabolized into dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase. We aimed to develop a LC-MS/MS method to quantify EETs and DHETs in human plasma and monitored their levels during vascular endothelial stimulation. Plasma samples, collected from 14 healthy and five hypertensive subjects at baseline and during radial artery endothelium-dependent flow-mediated dilatation, were spiked with internal standards. Lipids were then extracted by a modified Bligh and Dyer method and saponified to release bound EETs and DHETs. Samples were purified by a second liquid-liquid extraction and analyzed by LC-MS/MS. The assay allowed identification of (±)8(9)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-EET); (±)11(12)-epoxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-EET); (±)14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-EET); (±)8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-DHET); (±)11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-DHET); and (±)14,15-dihydroxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-DHET). (±)5(6)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (5,6-EET) was virtually undetectable due to its chemical instability. The limits of quantification were 0.25 ng/mL for DHETs and 0.5 ng/mL for EETs. Intra- and inter-assay variations ranged from 1.6 to 13.2%. Heating induced a similar increase in 8,9-EET, 11,12-EET, and 14,15-EET levels and in corresponding DHET levels in healthy but not in hypertensive subjects. We validated a sensitive LC-MS/MS method for measuring simultaneously plasma EET and DHET regioisomers in human plasma and showed its interest for assessing endothelial function.

The antiapoptotic effect of remifentanil on the immature mouse brain: an ex vivo study.

BACKGROUND: The use of remifentanil in a context of potential prematurity led us to explore ex vivo the opioid effects on the immature mouse brain. Remifentanil enhances medullary glutamatergic N-methyl-D-aspartate (NMDA) receptor activity. Furthermore, in neonatal mouse cortex, NMDA was previously shown to exert either excitotoxic or antiapoptotic effects depending on the cortical layers. With the use of a model of acute cultured brain slices, we evaluated the potential necrotic and apoptotic effects of remifentanil, alone or associated with its glycine vehicle (commercial preparation of remifentanil, C.P. remifentanil), on the immature brain. METHODS: Cerebral slices from postnatal day 2 mice were treated up to 5 hours with the different compounds, incubated alone or in the presence of NMDA. The necrotic effect was studied by measuring lactate dehydrogenase activity and 7-Aminoactinomycin D labeling. Apoptotic death was evaluated by measurement of caspase-3 activity and cleaved caspase-3 protein levels, using Western blot and immunohistochemistry. Extrinsic and intrinsic apoptotic pathways were investigated by measuring caspase-8, caspase-9 activities, Bax protein levels, and mitochondrial integrity. RESULTS: C.P. remifentanil was ineffective on necrotic death, whereas it significantly reduced caspase-3 activity and cortical cleaved caspase-3 levels. C.P. remifentanil inhibited cortical Bax protein expression, caspase-9 activity, and preserved mitochondrial integrity, whereas it had no effect on caspase-8 activity. Its action targeted the neocortex superficial layers, and it was reversed by the opioid receptors antagonist naloxone and the NMDA antagonist MK801. Remifentanil and glycine acted synergistically to inhibit apoptotic death. In addition, C.P. remifentanil enhanced the antiapoptotic effect of NMDA, whereas it did not improve NMDA excitotoxicity in brain slices. CONCLUSION: The present data indicate that at a supraclinical concentration C.P. remifentanil had no pronecrotic effect but exerted ex vivo antiapoptotic action on the immature mouse brain, involving the opioid and NMDA receptors, and the mitochondrial-dependent apoptotic pathway. Assessment of the impact of the antiapoptotic effect of remifentanil in in vivo neonatal mouse models of brain injury will also be essential to measure its consequences on the developing brain.

Invasive Geotrichum clavatum fungal infection in an acute myeloid leukaemia patient: a case report and review.

Invasive Geotrichum clavatum fungal infections are extremely rare and unusual, occurring nearly exclusively in patients experiencing prolonged neutropenia during the treatment for acute myeloid leukaemia. Several groups of cases of fatal G. clavatum infection were reported in France between 2011 and 2012, but the ecological niche has not yet been identified. We report a case of a 32-year-old patient with acute myeloid leukaemia who developed G. clavatum sepsis with primary peritonitis, hepatic nodular lesions, and multivisceral failure during aplasia after induction followed by salvage chemotherapy. He was treated with voriconazole and is still alive 1 year after with controlled disease. We then discuss the epidemiological, clinical, and therapeutic features of these serious fungal infections compared to the published data.

Metoclopramide stimulates catecholamine- and granin-derived peptide secretion from pheochromocytoma cells through activation of serotonin type 4 (5-HT4) receptors.

UNLABELLED: The gastroprokinetic agent metoclopramide is known to stimulate catecholamine secretion from pheochromocytomas. The aim of the study was to investigate the mechanism of action of metoclopramide and expression of serotonin type 4 (5-HT(4)) receptors in pheochromocytoma tissues. Tissue explants, obtained from 18 pheochromocytomas including the tumor removed from a 46-year-old female patient who experienced life-threatening hypertension crisis after metoclopramide administration and 17 additional pheochromocytomas (9 benign and 8 malignant) were studied. Cultured pheochromocytoma cells derived from the patient who previously received metoclopramide were incubated with metoclopramide and various 5-HT(4) receptor ligands. In addition, total mRNAs were extracted from all the 18 tumors. Catecholamine- and granin-derived peptide concentrations were measured in pheochromocytoma cell incubation medium by HPLC and radioimmunological assays. In addition, expression of 5-HT(4) receptor mRNAs in the 18 pheochromocytomas was investigated by the use of reverse transcriptase-PCR. RESULTS: Metoclopramide and the 5-HT(4) receptor agonist cisapride were found to activate catecholamine- and granin-derived peptide secretions by cultured tumor cells. Metoclopramide- and cisapride-evoked catecholamine- and granin-derived peptide productions were inhibited by the 5-HT(4) receptor antagonist GR 113808. 5-HT(4) receptor mRNAs were detected in the patient's tumor and the series of 17 additional pheochromocytomas. This study shows that pheochromocytomas express functional 5-HT(4) receptors that are responsible for the stimulatory action of metoclopramide on catecholamine- and granin-derived peptide secretion. All 5-HT(4) receptor agonists must therefore be contraindicated in patients with proven or suspected pheochromocytoma.

Beneficial Effects of Remifentanil Against Excitotoxic Brain Damage in Newborn Mice.

Background: Remifentanil, a synthetic opioid used for analgesia during cesarean sections, has been shown in ex vivo experiments to exert anti-apoptotic activity on immature mice brains. The present study aimed to characterize the impact of remifentanil on brain lesions using an in vivo model of excitotoxic neonatal brain injury. Methods: Postnatal day 2 (P2) mice received three intraperitoneal injections of remifentanil (500 ng/g over a 10-min period) or saline just before an intracortical injection of ibotenate (10 µg). Cerebral reactive oxygen species (ROS) production, cell death, in situ labeling of cortical caspase activity, astrogliosis, inflammation mediators, and lesion size were determined at various time points after ibotenate injection. Finally, behavioral tests were performed until P18. Results: In the injured neonatal brain, remifentanil significantly decreased ROS production, cortical caspase activity, DNA fragmentation, interleukin-1ß levels, and reactive astrogliosis. At P7, the sizes of the ibotenate-induced lesions were significantly reduced by remifentanil treatment. Performance on negative geotaxis (P6-8) and grasping reflex (P10-12) tests was improved in the remifentanil group. At P18, a sex specificity was noticed; remifentanil-treated females spent more time in the open field center than did the controls, suggesting less anxiety in young female mice. Conclusions: In vivo exposure to remifentanil exerts a beneficial effect against excitotoxicity on the developing mouse brain, which is associated with a reduction in the size of ibotenate-induced brain lesion as well as prevention of some behavioral deficits in young mice. The long-term effect of neonatal exposure to remifentanil should be investigated.

Impact of CYP2C19 genetic polymorphisms on voriconazole dosing and exposure in adult patients with invasive fungal infections.

Voriconazole (VCZ) use is limited by its narrow therapeutic range and significant interpatient variability in exposure. This study aimed to assess (i) the impact of CYP2C19 genotype on VCZ exposure and (ii) the doses required to achieve the therapeutic range in adult patients with invasive fungal infections (IFIs). Therapeutic drug monitoring (TDM) of VCZ, based on trough concentration measurement, and CYP2C19 genotyping were used to guide VCZ dosing in Caucasian patients with IFIs. The two common polymorphisms in Caucasians (CYP2C19*2 and *17), associated with decreased or increased CYP2C19 activity, respectively, were correlated with the daily VCZ dose, pharmacokinetic parameters and concentration-to-dose ratio. In total, 111 trough concentration measurements from 35 genotyped patients were analysed using linear mixed-effect models. The mean VCZ doses required to achieve target concentrations were significantly higher in CYP2C19*17 carriers compared with CYP2C19*1/*1 individuals (P<0.001): 2.57±0.25mg/kg twice daily in CYP2C19*1/*1 patients versus 3.94±0.39mg/kg and 6.75±0.54mg/kg in *1/*17 and *17/*17 patients, respectively. In addition, exposure to VCZ correlated with the CYP2C19*17 variant. Indices of exposure for CYP2C19*2 carriers were in line with the functional effect of this polymorphism compared with CYP2C19*1/*1 individuals, however comparisons of doses required to achieve target concentrations were not statistically different. The CYP2C19*17 allele predicted both VCZ exposure and dose required to achieve effective and non-toxic concentrations. CYP2C19 genotyping appears useful to guide VCZ initial dosing when coupled with TDM and to explain subtherapeutic concentrations frequently observed in clinical practice.

Severe acute poisoning with homemade Aconitum napellus capsules: toxicokinetic and clinical data.

Aconitum napellus is an extremely dangerous plant that contains various toxic diterpenoid alkaloids, mainly aconitine primarily concentrated in the roots. We report a case of acute intoxication of a 21-year-old man admitted to our Emergency Department after the ingestion, in order to sleep, of three homemade Aconitum napellus capsules. Capsules were measured to contain 237 mg of root and 19 microg of aconitine. The patient experienced the first symptoms on wakening 5 hours later with generalized paresthesia, nausea, diarrhea, vertigo, thoracic pain dyspnea, and dyschromatopsia. At admission, 7 hours after intake electrocardiographic analysis showed a sinusal bradycardia with polymorphic and bigeminal ventricular extrasystolia. Cardiovascular and neurological symptoms disappeared, respectively within 11 and 13 hours of ingestion. The patient was discharged from the ICU on day 2. Plasmatic concentrations at H7, H9, H14 H19, and after ingestion were, respectively, of 1.75, 0.75, 0.35, and 0.02 ng/mL. The calculated half-life of aconitine was 3 hours. To our knowledge, this is the first reported case with an aconitine toxicokinetic-effect relationship. The authors stress that clinicians must be aware of possible occurrence of acute poisoning with Aconitum napellus in European countries and in the United States as herbal medicine is becoming increasingly popular.

Protective effect of mycophenolate mofetil on endothelial function in an aortic allograft model.

BACKGROUND: Whether mycophenolate mofetil (MMF) can prevent the vascular endothelial dysfunction related to the administration of calcineurin inhibitor after organ transplantation remains unknown. METHODS: Four groups of Lewis rats, grafted with Brown Norway donor aortic abdominal allograft, received since the transplantation cyclosporine A (CsA, 5 mg/kg/day), MMF (40 mg/kg/day), CsA+MMF, or vehicle (control) for 2 weeks. RESULTS: Fifteen days after transplantation, all immunosuppressive regimens were equally effective in preventing graft rejection. When compared with control rats, the endothelium-dependent relaxation to acetylcholine was reduced, and the vasoconstrictor effect of phenylephrine was enhanced in thoracic aorta of CsA-treated rats but not in rats treated with MMF alone or combined with CsA without difference for the endothelium-independent relaxation to sodium nitroprusside. The relaxation to acetylcholine was abolished by the nitric oxide (NO)-synthase inhibitor N-nitro-l-arginine in all groups. Moreover, the endothelial NO-synthase protein dimer:monomer ratio in the thoracic aorta and the plasma nitrites concentrations, an indicator of NO availability, were decreased in CsA-treated rats but not in rats treated with MMF alone or combined with CsA. CONCLUSIONS: This study demonstrates that MMF prevents systemic endothelial dysfunction and the enhanced sensitivity to vasoconstrictors related to CsA administration in a rat allograft aortic model through an increase in NO availability related to the improvement of endothelial NO-synthase functionality.

Cost-effectiveness analysis of individualized mycophenolate mofetil dosing in kidney transplant patients in the APOMYGRE trial.

BACKGROUND: In the prospective, randomized, multicenter APOMYGRE trial conducted in France, concentration-controlled mycophenolate mofetil (MMF) dosing based on mycophenolic acid (MPA) exposure significantly reduced the treatment failure and acute rejection during the first posttransplantation year compared with fixed-dose MMF. This analysis investigated the cost effectiveness of dose individualization. METHOD: The study included 65 patients per group (intent-to-treat population). Treatment failure (primary efficacy endpoint) was defined as death, graft loss, acute rejection, or MMF discontinuation because of adverse effects. Data on hospitalizations, drugs prescribed, physicians' fees, laboratory expenses, ambulatory visits, and transportation were retrieved. Costs were calculated from the French National Health System perspective. RESULTS: The mean (95% confidence interval) total yearly cost per patient was Euro 47,477 (Euro 43,933; Euro 51,020) in the concentration-controlled group and Euro 46,783 ( Euro 44,152; Euro 49,414) in the fixed-dose group (P=0.7). The observed incremental cost-effectiveness ratio was Euro 3757 per treatment failure (Purchasing Power Parities United States/France: $4129). Hospitalization and drug costs accounted for approximately 50% and 25% of total costs, respectively. The cost for MPA area under the concentration-time curve and dose calculation was Euro 452 per patient, less than 1% of the total cost. CONCLUSION: In the APOMYGRE trial, therapeutic MPA monitoring using a limited sampling strategy reduced the risk of treatment failure and acute rejection in renal allograft recipients during the first 12 months posttransplantation, at neutral cost.

Comparative effects of ivabradine, a selective heart rate-lowering agent, and propranolol on systemic and cardiac haemodynamics at rest and during exercise.

AIM: To compare in humans the effects of ivabradine and propranolol on cardiac and systemic haemodynamics at rest, during tilt and exercise. METHODS: Nine healthy volunteers randomly received single oral doses of ivabradine (Iva, 30 mg), propranolol (Propra, 40 mg) or placebo (Plac) during a double-blind cross-over study. Doses were selected to be equipotent in heart rate (HR) reduction. HR, systolic and diastolic blood pressure (SBP, DBP), cardiac index (CI, bioimpedance), rate pressure product (RPP), plasma epinephrine (E) and norepinephrine (NE), were measured at rest at baseline, before and after two tilt and exercise tests, started 2 and 5 h after drug intake. Heart rate variability (low to high frequency ratio LF/HF) was evaluated at rest and at 5 th minute of tilt. RESULTS: At rest, HR and RPP decreased similarly with Iva and Propra (both P < 0.01). During tilt, HR increased less with Iva than Propra (P < 0.01), LF/HF decreased after Iva (P < 0.03), SBP and mean blood pressure decreased after Propra (both P < 0.01), RPP decreased similarly after Iva and Propra (both P < 0.01) and CI decreased to a greater extent with Propra than with Iva or Plac (both P < 0.04). During exercise, Iva and Propra similarly decreased HR (both P < 0.01) and RPP (P < 0.01). CONCLUSIONS: These results demonstrate that for a similar decrease in HR at rest and during sympathetic stimulation, acute administration of ivabradine, a selective heart rate-lowering agent, decreased myocardial oxygen demand to the same extent as a reference beta-blocker, propranolol, but without evidence of depressant effect on cardiac function.

Influence of vascular dimension on gender difference in flow-dependent dilatation of peripheral conduit arteries.

To assess the influence of initial diameter on the gender difference in flow-dependent dilatation (FDD) of the conduit artery, we measured radial artery internal diameter (echotracking), flow (Doppler) and total blood viscosity in 24 healthy (25 +/- 0.8 yr) men and women during reactive hyperemia (RH) and during a gradual hand skin heating (SH). At baseline, mean diameter (men, 2.76 +/- 0.09 vs. women, 2.32 +/- 0.07 mm, P < 0.05), flow (men, 21 +/- 4 vs. women, 10 +/- 1 ml/min, P < 0.05), and blood viscosity (men, 4.13 +/- 0.07 vs. women, 3.92 +/- 0.13 cP, P < 0.05) were higher in men but mean shear stress (MSS) was not different between groups. During RH, the percent increase in diameter was lower in men (men, 9 +/- 1 vs. women, 13 +/- 1%, P < 0.05). This difference was suppressed after correction for baseline diameter. During SH, the increase in diameter with flow was higher in women (P < 0.01). However, the increase in MSS was higher in women because of their smaller diameter at each level of flow (P < 0.01) and there was no difference between groups for the increase in diameter at each level of MSS. These results demonstrate in a direct manner that initial diameter influences the magnitude of FDD of conduit arteries in humans by modifying the value of the arterial wall shear stress at each level of flow and support the interest of the heating method in presence of heterogeneous groups.